Parvovirus b19 DNA is commonly harboured in human skin.

BACKGROUND: Parvovirus B19 is the aetiological agent of erythema infectiosum. The presence of B19 DNA in lesional skin of other cutaneous manifestations has frequently been reported although there is disagreement on the role of the B19 virus in tissues. OBJECTIVES: To investigate the presence of B19 DNA (1) in skin lesions of patients with a described B19-related disease, (2) in skin lesions of B19-unrelated diseases and (3) in healthy skin. METHODS: A total of 121 skin samples were examined for the presence of B19 DNA by PCR assays and peptide-nucleic-acid-based in situ hybridisation techniques. RESULTS: B19 DNA was detected in 11/38 (28.9%) pityriasis lichenoides, 8/30 (26.7%) melanocytic naevi, 5/29 (17.2%) primary melanomas and 6/24 (25.0%) healthy skin biopsies. A difference in B19 DNA prevalence was observed in specimens grouped according to age, irrespective of pathologies. CONCLUSIONS: B19 DNA can be found in skin tissues of patients with pityriasis lichenoides as well as in lesions not related to B19 infection and in healthy controls. B19 DNA can be detected in skin of young subjects in a significantly high rate compared to adults, suggesting that viral persistence may be the usual outcome after primary infection.

The density of anthropogenic features explains seasonal and behaviour-based functional responses in selection of linear features by a social predator.

Anthropogenic linear features facilitate access and travel efficiency for predators, and can influence predator distribution and encounter rates with prey. We used GPS collar data from eight wolf packs and characteristics of seismic lines to investigate whether ease-of-travel or access to areas presumed to be preferred by prey best explained seasonal selection patterns of wolves near seismic lines, and whether the density of anthropogenic features led to functional responses in habitat selection. At a broad scale, wolves showed evidence of habitat-driven functional responses by exhibiting greater selection for areas near low-vegetation height seismic lines in areas with low densities of anthropogenic features. We highlight the importance of considering landscape heterogeneity and habitat characteristics, and the functional response in habitat selection when investigating seasonal behaviour-based selection patterns. Our results support behaviour in line with search for primary prey during summer and fall, and ease-of-travel during spring, while patterns of selection during winter aligned best with ease-of-travel for the less-industrialized foothills landscape, and with search for primary prey in the more-industrialized boreal landscape. These results highlight that time-sensitive restoration actions on anthropogenic features can affect the probability of overlap between predators and threatened prey within different landscapes.

Diagnosis of fetal parvovirus B19 infection: value of virological assays in fetal specimens.

OBJECTIVE: The purpose of our work was to examine the most reliable laboratory diagnosis of fetal parvovirus B19 infection in hydropic fetuses by evaluating the most appropriate clinical sample and laboratory test. DESIGN: B19 DNA detection in fetal samples and serological signs of B19 infection in the respective mothers. Samples collected between January 2000 and July 2008. SETTING: Microbiology, University of Bologna, Bologna, Italy. SAMPLES: One hundred thirty-five fetal samples (58 fetal cord blood and 77 amniotic fluid samples) and 109 serum samples collected from 109 pregnant women. METHODS: Validated and certified in situ hybridisation assay (ISH) and polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) were performed on fetal samples to detect B19 DNA. B19-specific antibodies were investigated in maternal serum samples by a commercial enzyme immunoassay. MAIN OUTCOME MEASURES: Parvovirus B19 DNA detection in fetal specimens was analysed in relation to maternal serological signs of infection. RESULTS: Parvovirus B19 DNA was detected in 22.41% of fetal cord blood and 36.36% of amniotic fluid samples. A statistically significant difference was found between DNA detection by ISH (23.70%) and PCR-ELISA (14.81%) (P= 0.004). Only 11.76% of fetuses with virological diagnosis of B19 infection were from women with serological signs of acute/recent B19 infection. CONCLUSIONS: Diagnosis of fetal parvovirus B19 infection cannot always rely on maternal serological investigations but rather on the virological analysis of fetal samples. Both fetal cord blood and amniotic fluid samples are suitable for diagnosis, but the detection of B19 DNA in the cells of amniotic fluid samples by ISH proved to be the most reliable diagnostic system.

Multiplex chemiluminescence microscope imaging of P16(INK4A) and HPV DNA as biomarker of cervical neoplasia.

Classification of cervical intraepithelial neoplasia (CIN) lesions in low-grade (CIN1) or high-grade (CIN2-3) ones is crucial for optimal patient management, but current histological diagnosis on bioptic samples is often hampered by inter-observer variability. To allow objective classification, we have exploited the peculiar characteristics of chemiluminescence detection, such as high sensitivity and easy quantification of the luminescence signal, to perform sequentially in the same tissue section both an immunohistochemical quantitative detection of p16(INK4A) (a protein marker of high-grade CIN lesions) and an in situ hybridization for human papillomavirus (generally accepted as a necessary but insufficient cause of cervical carcinoma). Different label enzymes (alkaline phosphatase and horseradish peroxidase) were employed in order to avoid any interference between the two assays, and quantitative chemiluminescence image analysis was used to obtain objective evaluation of sample positivity. The multiplexed method allowed detection of two complementary biomarkers and provided discrimination between different lesions (non-neoplastic, low-grade and high-grade CIN). This assay might thus represent an accurate and objective diagnostic test providing important information for counseling, selection of therapy and follow up after surgical treatment.

Rapid diagnosis of viral infections by an alkaline phosphatase immunocytochemical method.

The use of alkaline phosphatase immunocytochemical staining was explored for the rapid diagnosis of poliovirus, adenovirus, herpes simplex virus and cytomegalovirus infections in cell cultures. In this test, viral antigens treated with their relative antibody were incubated with alkaline phosphatase-labelled antisera. The enzyme label was developed with a naphthol salt in the presence of a diazonium salt (Fast Blue) in order to obtain a blue coloured precipitate at the site of the enzyme. It is suggested that this immunocytochemical technique is valuable in the detection of viral infections and would be an appropriate test to use when rapid diagnosis is required.

A hybrido-immunocytochemical assay for the in situ detection of cytomegalovirus DNA using digoxigenin-labeled probes.

A non-radioactive hybrido-immunocytochemical assay for the detection of cytomegalovirus (CMV) DNA in infected cells was developed. Two different DNA fragments belonging to the repeated sequences of CMV genome were used to construct the hybridization probe. The probe was constructed by incorporating deoxyuridine triphosphate labeled with digoxigenin. The in situ hybridized CMV DNA probe was immunocytochemically visualized by anti-digoxigenin. Fab fragments labeled with alkaline phosphatase. This procedure permitted the DNA detection, in the nuclei of infected cells fixed at 48 h after infection, of the Towne CMV reference strain and 21 different laboratory-isolated CMV strains. Our assay demonstrated a high specificity, sensitivity and reproducibility.

Co-localization of two different viral genomes in the same sample by double-chemiluminescence in situ hybridization.

A double-chemiluminescence in situ hybridization has been developed that combines the advantages of chemiluminescence with the detection of two different viral DNAs, i.e., herpes simplex virus (HSV) DNA and cytomegalovirus (CMV) DNA, in infected cells in the same specimen. For the simultaneous detection of these two different viral DNAs, we used a biotinylated HSV DNA probe, which can be visualized by a streptavidin-horseradish peroxidase (HRP) complex amplified with biotinyl tyramide. This probe was followed by the use of a luminol-based chemiluminescent substrate for HRP and a digoxigenin-labeled CMV DNA probe visualized by antidigoxigenin Fab fragments conjugated with alkaline phosphatase (AP). This is followed by the detection with a dioxetane phosphate derivate as chemiluminescent substrate for AP. Since the final product of both chemiluminescent reactions was light emission, sequential images for the two hybridizations were taken and analyzed using a high-performance luminograph connected to an optical microscope and to a personal computer for image analysis. Positive signals for the presence of both HSV DNA and CMV DNA were noticed in infected cells in the same specimen with a sharp localization, absence of cross reactions and absence of background.

Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes.

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.

Sensitive chemiluminescence in situ hybridization for the detection of human papillomavirus genomes in biopsy specimens.

We developed a sensitive chemiluminescence in situ hybridization assay for detection of human papillomavirus (HPV) DNA for objective and semiquantitative evaluation of the results. The hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes, visualized with alkaline phosphatase as the revealing enzyme and a highly sensitive 1,2 dioxetane phosphate as chemiluminescent substrate. The light emitted from the hybridized probes was detected, analyzed, and measured using a high-performance, low light-level imaging luminograph connected to an optical microscope and to a personal computer for quantification of the photon fluxes and for image analysis. The system operated in consecutive steps: First, hybridized specimens were recorded in transmitted light. Then the net luminescent signal was recorded, and then an overlay of the two images provided by the transmitted light and by the luminescent signal allowed the spatial distribution of the target DNA to be localized, measured, and evaluated. Biopsy specimens from different pathological conditions associated with HPV, which had previously been proved positive for HPV DNA with the polymerase chain reaction (PCR), were analysed. The chemiluminescence in situ hybridization proved sensitive and specific with digoxigenin-, biotin-, or fluorescein-labeled probes, and provided an objective evaluation of the results. The results obtained with chemiluminescence in situ hybridization were also compared with results obtained with in situ hybridization with colorimetric detection, with good concordance of the data. Chemiluminescence in situ hybridization therefore offers the possibility of detecting HPV DNA with great sensitivity in biopsy specimens. Moreover, the images of the samples, stored in the computer, are a permanent record of the reaction and can also be sent for evaluation or comparison to other laboratories using computer networks.

Stimulating effect of heat shock on the early stage of human cytomegalovirus replication cycle.

The effect of mild heat shock on the replication of human cytomegalovirus (HCMV) was studied in human embryo fibroblasts. Treatment of cell cultures at 44 degrees C for 10 min just before infection or at 24 h post infection (p.i.) shortened HCMV eclipse period and enhanced viral replication, while heat shock performed at 48 h p.i. had no effect on the HCMV replication cycle. Study of HCMV-induced early and late antigens confirmed that the cellular response to heat shock influences HCMV replication in the early stage of the viral replication cycle.

Identification of an immunodominant peptide in the parvovirus B19 VP1 unique region able to elicit a long-lasting immune response in humans.

The immune response against parvovirus B19 is mainly directed against the two structural proteins, VP1 and VP2. The amino terminal half of the VP1 unique region has been shown to elicit a dominant immune response in humans, more effective than other linear epitopes and also it has been seen to contain significant neutralizing linear epitopes. Three overlapping recombinant peptides corresponding to amino acids 2-40 (VP1-A), amino acids 32-71 (VP1-B), and amino acids 60-100 (VP1-C) of the VP1 unique region were produced by a procaryotic expression system. These peptides were used as antigens in a Western blot assay to detect specific immunoglobulin G (IgG) in serum samples from blood donors of different age groups with documented signs of a past B19 infection. Fragment VP1-C appeared significantly immunodominant over the other peptides, reacting with specific IgG in 86% of serum samples. The fragment VP1-C corresponds to a sequence with a known neutralizing activity and seems able to elicit a long-lasting immune response because specific IgG were present in blood donors of all age groups. VP1-C would therefore appear to be an attractive candidate as a component of a subunit vaccine.

Efficient treatment of paraffin-embedded cervical tissue for HPV DNA testing by HC-II and PCR assays.

BACKGROUND AND OBJECTIVES: High-risk human papillomaviruses (HR-HPVs) are the primary cause of cervical cancer. In order to meet with clinical requirements, a direct capture test with signal amplification (HC-II), able to detect the 13 prevalent HR-HPVs, has been developed and validated for cytological specimens. STUDY DESIGN: the use of HC-II assay with formaldehyde-fixed paraffin-embedded cervical biopsies, for retrospective studies or to support histological findings, was investigated by analysing three different sample treatments. The efficacy of this test was compared with a reference PCR-ELISA, using MY09/11 and GP5+/6+ consensus primers and the use of a single extraction method for both HC-II and PCR-ELISA assays was validated. RESULTS: protease treatment of dewaxed biopsy sections allowed an optimal performance of HC-II and has also been validated for PCR-ELISA. Overall, on the analysis of 50 cervical samples HC-II and PCR-ELISA assays showed a high concordance (K=0.80). Compared with PCR-ELISA, the HC-II had a sensitivity of 86.4% and a specificity of 92.9%. The results showed that short amplimers are necessary for a sensitive PCR-ELISA from formaldehyde-fixed paraffin-embedded biopsies, while HC-II showed a relatively low sensitivity for HPV18 within the HR probe pool. CONCLUSIONS: HC-II can be a valid tool for the diagnosis of HPV infection in biopsy material. The possibility to use the same specimen preparation material for both HC-II and PCR-ELISA allows HC-II positive specimens to be further processed by PCR-ELISA if specific genotyping is needed.

Chemiluminescence: a sensitive detection system in in situ hybridization.

Chemiluminescence is the light emission produced by a chemical reaction in which chemically excited molecules decay to the ground state. The phenomenon is utilized in various analytical techniques in which small amounts of analytes or enzymes can be detected and quantified by measurement of the light emitted by bio- or chemiluminescent reactions. Recently chemiluminescence has been proposed as a valid alternative to radioactive or colorimetric methods in in situ hybridization assays, in which target nucleic acids are localized by labeled probes inside individual cells with the preservation of cell morphology. Chemiluminescence in situ hybridization is performed using probes that are detected using enzymes with their appropriate chemiluminescent substrates. The luminescent signal from the hybrid formation is detected, analysed and measured with a high performance low light level imaging apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Generally, the instrumental system to detect positive signals after in situ hybridization operates in three steps: firstly tissue structures and cells are recorded in transmitted light then the luminescent signal is measured with an optimized photon accumulation; and then, after a computer elaboration of the luminescent signal with pseudocolors corresponding to the light intensity, an overlay of the two images on the screen provided by the transmitted light and by the luminescent signal allows the spatial distribution of the labeled probe to be localized and evaluated. The main advantages of chemiluminescence in situ hybridization are mainly the sensitivity, the quantification of the data, the objectivity of the evaluation and the digital imaging of the results. The chemiluminescence in situ hybridization assay, which can be applied to cell smears, archival frozen and paraffin embedded tissue samples, can be a useful tool for a sensitive and specific diagnosis of viral infections and for the detection and study of specific genic sequences inside the cells. The use of the chemiluminescent in situ hybridization assay is also promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.

Rapid detection of antibodies against cytomegalovirus induced immediate early and early antigens by an enzyme linked immunosorbent assay.

An enzyme linked immunosorbent assay (ELISA) for detecting antibodies against cytomegalovirus induced immediate early antigens and early antigens was developed using purified nuclear antigens and was compared with the indirect immunofluorescence test. The tests were comparable in their ability to detect positive and negative sera, and antibody titres determined by both assays were similar. The use of ELISA for the detection of antibodies against cytomegalovirus induced immediate early and early antigens is advocated in diagnostic and research laboratories.

Alkaline phosphatase immunoenzymatic staining for detection of antigens induced by cytomegalovirus.

An indirect alkaline phosphatase immunoenzymatic staining method was developed to localise antigens in human embryo fibroblasts that have been induced by cytomegalovirus. The enzyme label was developed with a naphthol salt and fast blue to obtain a bright blue staining of the antigens that could be clearly visualised under an ordinary light microscope. The procedure is rapid, sensitive, and specific and can be used in diagnostic laboratories to detect active infection caused by cytomegalovirus.

Impairment of cytomegalovirus and host balance in elderly subjects.

The titres of IgG antibody against "late antigens", "immediate early antigens", and "early antigens" induced by cytomegalovirus (CMV) and IgM antibody against "late antigens" induced by CMV were analysed in 67 geriatric subjects by immunocytochemical techniques. The titres obtained were compared with those of an adult control population. Significantly increased titres of IgG antibody against induced antigens and a significant increase in CMV reactivated infections occurred in the elderly compared with control subjects. These findings indicate that the CMV and host balance in the elderly is disturbed, leading to activation of the CMV latent carrier state that follows primary CMV infection.

Rapid diagnosis of cytomegalovirus encephalitis in patients with AIDS using in situ hybridisation.

AIMS: To evaluate the presence of cytomegalovirus (CMV) DNA in the cerebrospinal fluid of patients with AIDS and suspected viral encephalitis using an in situ hybridisation assay with digoxigenin labelled CMV DNA probes. METHODS: The presence of CMV DNA was evaluated in cerebrospinal fluid cells of 10 patients with AIDS using in situ hybridisation. The positivity of CMV DNA was confirmed by the presence of CMV induced antigens in the same specimens. The presence of CMV DNA and CMV induced antigens was also analysed in peripheral blood leucocytes. The time required to perform the in situ hybridisation assay was about eight hours. RESULTS: The in situ hybridisation assay was sensitive, specific, and provided good resolution. Six patients proved positive for the presence of CMV DNA in CSF cells and all six also proved positive for CMV DNA in blood leucocytes. Of the six CMV positive patients, five were treated with specific antiviral drugs: of these, one died during the treatment while four clinically recovered after one month of treatment. CONCLUSIONS: The in situ hybridisation assay using digoxigenin labelled CMV DNA probes can be used as a valuable diagnostic test for the detection of CMV DNA in the cerebrospinal fluid cells of patients with suspected CMV encephalitis and can therefore prompt adequate antiviral therapeutic intervention.

Distribution and viral load of type specific HPVs in different cervical lesions as detected by PCR-ELISA.

AIMS: To investigate the distribution and viral load of the most prevalent high risk human papillomavirus (HPV) types 16, 18, 31, 33, and 45 and low risk HPV types 6 and 11 in a variety of cervical lesions. METHODS: One hundred and seventy six cytological specimens from women with different cervical lesions were investigated. For an accurate standardisation of the sample, cervical cells were counted and a volume of the cell suspension processed by polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA). Semiquantitative determinations were achieved in relation to an external reference titration curve. RESULTS: HPV DNA was detected in 60.2% of the samples. HPV-16 was the prevalent genotype (57.6%), followed by HPV-33, HPV-31, HPV-6, HPV-18, and HPV-45. HPV-11 was not detected. HPV-16 showed a pronounced increase in prevalence with the evolution of cervical disease. Semiquantitative evaluation of the results showed that only HPV-16 DNA could reach very high values (> 1000 genome copies/cell) and a very high HPV-16 load correlated with the severity of cervical disease. CONCLUSIONS: Only HPV-16 load appears to be associated with the severity of cervical disease.

Evaluation of immunoassays for the detection and typing of PCR amplified human papillomavirus DNA.

AIMS: To evaluate different hybridisation techniques to detect and type human papillomavirus (HPV) DNAs amplified by consensus primer polymerase chain reaction (PCR) in biopsy and cytological specimens. METHODS: A hybrid capture-immunoassay in microtitre wells was performed to detect HPV sequences amplified by PCR and typed by specific oligoprobes. Consensus primers were used to amplify a sequence within the L1 open reading frame, and direct digoxigenin labelling of amplified products was performed during the amplification reaction. The amplified product was separately hybridised with six biotinylated type specific probes (HPV6, 11, 16, 18, 31, and 33); hybrids were then captured into streptavidin coated microtitre wells and detected by a spectrophotometer as an ELISA using antidigoxigenin Fab fragment labelled with peroxidase and a colorimetric substrate. The results were compared with the dot-blot immunoassay used to detect and type PCR amplified HPV DNA sequences. Consensus primers were used to generate the same unlabelled PCR product; digoxigenin labelled type specific probes for HPV6, 11, 16, 18, 31, and 33 were used and hybrids visualised by colorimetric immunoenzymatic reaction. Thirty nine biopsy specimens and 31 cytological samples were tested by the PCR-ELISA and by standard PCR followed by dot-blot hybridisation. RESULTS: The PCR-ELISA proved to be more sensitive than standard PCR with dot-blot hybridisation typing. All samples positive for HPV-DNA in standard PCR with dot-blot hybridisation method were confirmed positive by the PCR-ELISA assay; however, seven samples were positive only by PCR-ELISA. CONCLUSIONS: The PCR-ELISA assay, which can be performed in one day, is easily standardised and therefore seems to be a practical, sensitive, and reliable diagnostic tool for the detection and typing of HPV genomes in biopsy and in cytological specimens in the routine diagnostic laboratory.